The present invention relates to the general field of genetic engineering and is directed, in particular, to improvements in the coding sequence for foreign genes to be expressed in the cells of higher plants.
It is now possible reliably and repetitively to insert foreign genes into the germ line cells of higher plants, at least for certain species. A variety of techniques exist, notably Agrobacterium-mediated plant transformation and particle-mediated plant transformation, by which foreign genes can be introduced into the germ line plants in such a fashion that progeny of the plants will bear the gene of interest which is inserted. Accordingly, one area of research directed toward the creation of improved transgenic plants of potential commercial interest is in the insertion into plants of useful genes obtained from other species or classes of organisms so that the benefits of the gene product can be imbued into certain lines of higher plants. Examples of gene products in which effort has been directed toward their expression in plants cells include various toxins for control of insects, genes coding for various kinds of viral or other pathogen disease resistance, and genes coding for resistances to specific herbicides or antibiotics. In many of these cases the gene which is desired to be expressed in the plant cell comes from a procaryotic or viral organism. Some foreign genes may be from other species of plant or from other plants of the same species. When heterologous genes from these sources are inserted into plants, using promoters and expression cassettes which have been found operable and effective to express genes in plant cells, the results have been found to be sometimes uneven. There are apparent differences in either the transcription or translation levels of given coding sequences in plant tissues, even if the coding sequences are under the control of identical transcriptional promoters and terminators.
An example of this phenomenon has been found to occur with the gene for the delta-endotoxin crystal protein gene from the soil dwelling microorganism Bacillus thuringiensis (hereinafter referred to as the B.t. gene). A number of B.t. genes coding for homologous proteins have been cloned and sequenced by a variety of investigators throughout the world. Several of genetic constructs including one of the B.t. genes have been used to create chimeric plant expression gene constructions which are then transferred into the cells of plants. The various B.t. genes have been found to have significant differences in the DNA coding regions of the genes, although there is relatively high homology in the proteins for which they code. Nevertheless, the B.t. genes have characteristically been found to express relatively poorly in plant cells as compared to most other gene products which have been introduced into the cells of higher plants. The phenomenon of poor or low expression appears to have been experienced in all examples to date resulting from the introduction of native coding sequences for B.t. genes into plants, even though the expression cassettes and promoters and transcription terminators varied from experiment to experiment. One possible explanation for the observed phenomenon might be some feature of the native bacterial coding sequence itself.
As is known to all of ordinary skill in molecular biology, the genetic code of three nucleotide units, or codons, specifying particular amino acids, is degenerate. While a single amino acid is specified by each three nucleotide codon which makes up the genetic code found in DNA or RNA, because there are less amino acids possible than there are codon arrangements possible, most amino acids are specified by more than one codon sequence. For example, the amino acids serine, arginine, and leucine are all specified by any of six possible codons. It is thus possible to have nucleotide coding sequences for proteins which can differ significantly in their nucleotide sequence while specifying an identical amino acid sequence for the resultant protein.
The present invention is summarized as a method for constructing chimeric coding sequences for expression in plant cells in which the native coding sequence for a foreign gene to be expressed in plant cells is modified by substituting for the codons in the foreign coding region codons which are preferentially expressed in plants. The codons preferred for expression in plants are determined by analysis of the codon usage pattern of plant genes which are natively efficiently expressed in native plant tissues.
The present invention is further summarized in that a plant is engineered with a chimeric gene construct including a protein coding region constructed, and least in part, by oligonucleotide synthesis wherein the oligonucleotides are selected on the basis of preferred codon usage as determined by the usage of codons in genes which express well natively in plants.
It is an object of the present invention to enable the efficient construction of plant genes so as to obtain high steady-state levels of transcription and expression.
It is another object of the present invention to provide a B.t. gene construction which provides for high steady-state level of transcription and expression of the B.t. delta endotoxin protein in plant cells.
Other objects, advantages, and features of the present invention will become apparent from the following specification when taken in conjunction with the accompanying drawings.